We plan to determine the role, if any, of host cell protein synthesis in maintaining a suitable intracellular environment in growth of the protozoan parasite Toxoplasma gondii. Cellular protein synthesis will be inhibited in one of two ways. We will use as host cell a CHO mutant with a thermolabile tRNA synthetase. Upon incubation of this mutant at 40 degress, protein synthesis is rapidly inhibited. Alternatively, protein synthesis by host cell will be blocked by the irreversible antimetabolite muconomycin. We plan to assay the growth of T. gondii in several ways under conditions of blockage of host protein synthesis. For example, intracellular parasites will be counted by light microscopy, will be assayed for infectivity by a plaque assay, and will be assayed for biochemical activity both through the incorporation of radioactive uracil (specific for the parasite growth) and the incorporation of H3-leucine (autoradiography). Other experiments will examine the dependence of the intracellular parasite upon purine and pyrimidine biosynthesis by the host cell. In order to obtain unequivocal data, cell mutants with defined biochemical defects in biosynthesis will be used as hosts. In each case the utilization of C-14 labeled glucose to make the parasite nucleic acid bases will be examined. If the parasite uses pre-existing host cell nucleic acids as the source of the basis, they will not be labeled by glucose in the medium. On the other hand if de novo synthesis is carried out by the parasite, the radioactive atoms of 14C glucose should be found in the purines and the pyrimidines of the host cell.